SET1 and p300 act synergistically, through coupled histone modifications, in transcriptional activation by p53.

The H3K4me3 mark in chromatin is closely correlated with actively transcribed genes, although the mechanisms involved in its generation and function are not fully understood. In vitro studies with recombinant chromatin and purified human factors demonstrate a robust SET1 complex (SET1C)-mediated H3K4 trimethylation that is dependent upon p53- and p300-mediated H3 acetylation, a corresponding SET1C-mediated enhancement of p53- and p300-dependent transcription that reflects a primary effect of SET1C through H3K4 trimethylation, and direct SET1C-p53 and SET1C-p300 interactions indicative of a targeted recruitment mechanism. Complementary cell-based assays demonstrate a DNA-damage-induced p53-SET1C interaction, a corresponding enrichment of SET1C and H3K4me3 on a p53 target gene (p21/WAF1), and a corresponding codependency of H3K4 trimethylation and transcription upon p300 and SET1C. These results establish a mechanism in which SET1C and p300 act cooperatively, through direct interactions and coupled histone modifications, to facilitate the function of p53.

Gene-Specific H1 Eviction through a Transcriptional Activator→p300→NAP1→H1 Pathway.

Linker histone H1 has been correlated with transcriptional inhibition, but the mechanistic basis of the inhibition and its reversal during gene activation has remained enigmatic. We report that H1-compacted chromatin, reconstituted in vitro, blocks transcription by abrogating core histone modifications by p300 but not activator and p300 binding. Transcription from H1-bound chromatin is elicited by the H1 chaperone NAP1, which is recruited in a gene-specific manner through direct interactions with activator-bound p300 that facilitate core histone acetylation (by p300) and concomitant eviction of H1 and H2A-H2B. An analysis in B cells confirms the strong dependency on NAP1-mediated H1 eviction for induction of the silent CD40 gene and further demonstrates that H1 eviction, seeded by activator-p300-NAP1-H1 interactions, is propagated over a CCCTC-binding factor (CTCF)-demarcated region through a distinct mechanism that also involves NAP1. Our results confirm direct transcriptional inhibition by H1 and establish a gene-specific H1 eviction mechanism through an activator→p300→NAP1→H1 pathway.

Role for Dpy-30 in ES cell-fate specification by regulation of H3K4 methylation within bivalent domains.

Histone H3K4 methylation is associated with active genes and, along with H3K27 methylation, is part of a bivalent chromatin mark that typifies poised developmental genes in embryonic stem cells (ESCs). However, its functional roles in ESC maintenance and differentiation are not established. Here we show that mammalian Dpy-30, a core subunit of the SET1/MLL histone methyltransferase complexes, modulates H3K4 methylation in vitro, and directly regulates chromosomal H3K4 trimethylation (H3K4me3) throughout the mammalian genome. Depletion of Dpy-30 does not affect ESC self-renewal, but significantly alters the differentiation potential of ESCs, particularly along the neural lineage. The differentiation defect is accompanied by defects in gene induction and in H3K4 methylation at key developmental loci. Our results strongly indicate an essential functional role for Dpy-30 and SET1/MLL complex-mediated H3K4 methylation, as a component of the bivalent mark, at developmental genes during the ESC fate transitions.

A PRC2-Kdm5b axis sustains tumorigenicity of acute myeloid leukemia.

Acute myeloid leukemias (AMLs) with the NUP98-NSD1 or mixed lineage leukemia (MLL) rearrangement (MLL-r) share transcriptomic profiles associated with stemness-related gene signatures and display poor prognosis. The molecular underpinnings of AML aggressiveness and stemness remain far from clear. Studies with EZH2 enzymatic inhibitors show that polycomb repressive complex 2 (PRC2) is crucial for tumorigenicity in NUP98-NSD1+ AML, whereas transcriptomic analysis reveal that Kdm5b, a lysine demethylase gene carrying "bivalent" chromatin domains, is directly repressed by PRC2. While ectopic expression of Kdm5b suppressed AML growth, its depletion not only promoted tumorigenicity but also attenuated anti-AML effects of PRC2 inhibitors, demonstrating a PRC2-|Kdm5b axis for AML oncogenesis. Integrated RNA sequencing (RNA-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq), and Cleavage Under Targets & Release Using Nuclease (CUT&RUN) profiling also showed that Kdm5b directly binds and represses AML stemness genes. The anti-AML effect of Kdm5b relies on its chromatin association and/or scaffold functions rather than its demethylase activity. Collectively, this study describes a molecular axis that involves histone modifiers (PRC2-|Kdm5b) for sustaining AML oncogenesis.

Mediator subunit MED1 is required for E2A-PBX1-mediated oncogenic transcription and leukemic cell growth.

The chimeric transcription factor E2A-PBX1, containing the N-terminal activation domains of E2A fused to the C-terminal DNA-binding domain of PBX1, results in 5% of pediatric acute lymphoblastic leukemias (ALL). We recently have reported a mechanism for RUNX1-dependent recruitment of E2A-PBX1 to chromatin in pre-B leukemic cells; but the subsequent E2A-PBX1 functions through various coactivators and the general transcriptional machinery remain unclear. The Mediator complex plays a critical role in cell-specific gene activation by serving as a key coactivator for gene-specific transcription factors that facilitates their function through the RNA polymerase II transcriptional machinery, but whether Mediator contributes to aberrant expression of E2A-PBX1 target genes remains largely unexplored. Here we show that Mediator interacts directly with E2A-PBX1 through an interaction of the MED1 subunit with an E2A activation domain. Results of MED1 depletion by CRISPR/Cas9 further indicate that MED1 is specifically required for E2A-PBX1-dependent gene activation and leukemic cell growth. Integrated transcriptome and cistrome analyses identify pre-B cell receptor and cell cycle regulatory genes as direct cotargets of MED1 and E2A-PBX1. Notably, complementary biochemical analyses also demonstrate that recruitment of E2A-PBX1 to a target DNA template involves a direct interaction with DNA-bound RUNX1 that can be further stabilized by EBF1. These findings suggest that E2A-PBX1 interactions with RUNX1 and MED1/Mediator are of functional importance for both gene-specific transcriptional activation and maintenance of E2A-PBX1-driven leukemia. The MED1 dependency for E2A-PBX1-mediated gene activation and leukemogenesis may provide a potential therapeutic opportunity by targeting MED1 in E2A-PBX1+ pre-B leukemia.

Structural and Biochemical Characterization of Porcine Epidemic Diarrhea Virus Papain-Like Protease 2.

Coronaviral papain-like proteases (PLpros) are essential enzymes that mediate not only the proteolytic processes of viral polyproteins during virus replication but also the deubiquitination and deISGylation of cellular proteins that attenuate host innate immune responses. Therefore, PLpros are attractive targets for antiviral drug development. Here, we report the crystal structure of papain-like protease 2 (PLP2) of porcine epidemic diarrhea virus (PEDV) in complex with ubiquitin (Ub). The X-ray structural analyses reveal that PEDV PLP2 interacts with the Ub substrate mainly through the Ub core region and C-terminal tail. Mutations of Ub-interacting residues resulted in a moderately or completely abolished deubiquitinylating function of PEDV PLP2. In addition, our analyses also indicate that 2-residue-extended blocking loop 2 at the S4 subsite contributes to the substrate selectivity and binding affinity of PEDV PLP2. Furthermore, the PEDV PLP2 Glu99 residue, conserved in alphacoronavirus PLpros, was found to govern the preference of a positively charged P4 residue of peptidyl substrates. Collectively, our data provided structure-based information for the substrate binding and selectivity of PEDV PLP2. These findings may help us gain insights into the deubiquitinating (DUB) and proteolytic functions of PEDV PLP2 from a structural perspective. IMPORTANCE Current challenges in coronaviruses (CoVs) include a comprehensive understanding of the mechanistic effects of associated enzymes, including the 3C-like and papain-like proteases. We have previously reported that the PEDV PLP2 exhibits a broader substrate preference, superior DUB function, and inferior peptidase activity. However, the structural basis for these functions remains largely unclear. Here, we show the high-resolution X-ray crystal structure of PEDV PLP2 in complex with Ub. Integrated structural and biochemical analyses revealed that (i) three Ub core-interacting residues are essential for DUB function, (ii) 2-residue-elongated blocking loop 2 regulates substrate selectivity, and (iii) a conserved glutamate residue governs the substrate specificity of PEDV PLP2. Collectively, our findings provide not only structural insights into the catalytic mechanism of PEDV PLP2 but also a model for developing antiviral strategies.

Functions of paralogous RNA polymerase III subunits POLR3G and POLR3GL in mouse development.

Mammalian cells contain two isoforms of RNA polymerase III (Pol III) that differ in only a single subunit, with POLR3G in one form (Pol IIIα) and the related POLR3GL in the other form (Pol IIIß). Previous research indicates that POLR3G and POLR3GL are differentially expressed, with POLR3G expression being highly enriched in embryonic stem cells (ESCs) and tumor cells relative to the ubiquitously expressed POLR3GL. To date, the functional differences between these two subunits remain largely unexplored, especially in vivo. Here, we show that POLR3G and POLR3GL containing Pol III complexes bind the same target genes and assume the same functions both in vitro and in vivo and, to a significant degree, can compensate for each other in vivo. Notably, an observed defect in the differentiation ability of POLR3G knockout ESCs can be rescued by exogenous expression of POLR3GL. Moreover, whereas POLR3G knockout mice die at a very early embryonic stage, POLR3GL knockout mice complete embryonic development without noticeable defects but die at about 3 wk after birth with signs of both general growth defects and potential cerebellum-related neuronal defects. The different phenotypes of the knockout mice likely reflect differential expression levels of POLR3G and POLR3GL across developmental stages and between tissues and insufficient amounts of total Pol III in vivo.

Identification and Functional Analysis of an Epsilon Class Glutathione S-Transferase Gene Associated with α-Pinene Adaptation in Monochamus alternatus.

Alpha-pinene is one of the main defensive components in conifers. Monochamus alternatus (Coleoptera: Cerambycidae), a wood borer feeding on Pinaceae plants, relies on its detoxifying enzymes to resist the defensive terpenoids. Here, we assayed the peroxide level and GST activity of M. alternatus larvae treated with different concentrations of α-pinene. Meanwhile, a gst gene (MaGSTe3) was isolated and analyzed. We determined its expression level and verified its function. The results showed that α-pinene treatment led to membrane lipid peroxidation and thus increased the GST activity. Expression of MaGSTe3 was significantly upregulated in guts following exposure to α-pinene, which has a similar pattern with the malonaldehyde level. In vitro expression and disk diffusion assay showed that the MaGSTe3 protein had high antioxidant capacity. However, RNAi treatment of MaGSTe3 did not reduce the hydrogen peroxide and malonaldehyde levels, while GST activity was significantly reduced. These results suggested MaGSTe3 takes part in α-pinene adaptation, but it does not play a great role in the resistance of M. alternatus larvae to α-pinene.

E2A-PBX1 functions as a coactivator for RUNX1 in acute lymphoblastic leukemia.

E2A, a basic helix-loop-helix transcription factor, plays a crucial role in determining tissue-specific cell fate, including differentiation of B-cell lineages. In 5% of childhood acute lymphoblastic leukemia (ALL), the t(1,19) chromosomal translocation specifically targets the E2A gene and produces an oncogenic E2A-PBX1 fusion protein. Although previous studies have shown the oncogenic functions of E2A-PBX1 in cell and animal models, the E2A-PBX1-enforced cistrome, the E2A-PBX1 interactome, and related mechanisms underlying leukemogenesis remain unclear. Here, by unbiased genomic profiling approaches, we identify the direct target sites of E2A-PBX1 in t(1,19)-positive pre-B ALL cells and show that, compared with normal E2A, E2A-PBX1 preferentially binds to a subset of gene loci cobound by RUNX1 and gene-activating machineries (p300, MED1, and H3K27 acetylation). Using biochemical analyses, we further document a direct interaction of E2A-PBX1, through a region spanning the PBX1 homeodomain, with RUNX1. Our results also show that E2A-PBX1 binding to gene enhancers is dependent on the RUNX1 interaction but not the DNA-binding activity harbored within the PBX1 homeodomain of E2A-PBX1. Transcriptome analyses and cell transformation assays further establish a significant RUNX1 requirement for E2A-PBX1-mediated target gene activation and leukemogenesis. Notably, the RUNX1 locus itself is also directly activated by E2A-PBX1, indicating a multilayered interplay between E2A-PBX1 and RUNX1. Collectively, our study provides the first unbiased profiling of the E2A-PBX1 cistrome in pre-B ALL cells and reveals a previously unappreciated pathway in which E2A-PBX1 acts in concert with RUNX1 to enforce transcriptome alterations for the development of pre-B ALL.

Investigation of Cytotoxicity and Oxidative Stress Induced by the Pyrethroid Bioallethrin in Human Glioblastoma Cells: The Protective Effect of Vitamin E (VE) and Its Underlying Mechanism.

Bioallethrin belongs to the family of pyrethroid insecticides. Previous studies have shown that bioallethrin affected the function of muscarinic receptor and subsequently induced neurotoxicity in different brain models. Reactive oxygen species (ROS) are generated in the metabolic course of the human body, which can cause human damage when overactivated. However, whether bioallethrin evokes cytotoxicity through ROS signaling and whether the antioxidant Vitamin E (VE) protects these cytotoxic responses in human glial cell model are still elusive. This study investigated the effect of bioallethrin on cytotoxicity through ROS signaling and evaluated the protective effect of the antioxidant VE in DBTRG-05MG human glioblastoma cells. The cell counting kit-8 (CCK-8) was used to measure cell viability. Intracellular ROS and glutathione (GSH) levels were measured by a cellular assay kit. The levels of apoptosis- and antioxidant-related protein were analyzed by Western blotting. In DBTRG-05MG cells, bioallethrin (25-75 µM) concentration-dependently induced cytotoxicity by increasing ROS productions, decreasing GSH contents, and regulating protein expressions related to apoptosis or antioxidation. Furthermore, these cytotoxic effects were partially reversed by VE (20 µM) pretreatment. Together, VE partially lessened bioallethrin-induced apoptosis through oxidative stress in DBTRG-05MG cells. The data assist us in identifying the toxicological mechanism of bioallethrin and offer future development of the antioxidant VE to reduce brain damage caused by bioallethrin.

The A'-helix of CYP11A1 remodels mitochondrial cristae.

BACKGROUND: CYP11A1 is a protein located in the inner membrane of mitochondria catalyzing the first step of steroid synthesis. As a marker gene for steroid-producing cells, the abundance of CYP11A1 characterizes the extent of steroidogenic cell differentiation. Besides, the mitochondria of fully differentiated steroidogenic cells are specialized with tubulovesicular cristae. The participation of CYP11A1 in the change of mitochondrial structure and the differentiation of steroid-producing cells, however, has not been investigated. METHODS: We engineered nonsteroidogenic monkey kidney COS1 cells to express CYP11A1 upon doxycycline induction and examined the mitochondrial structure of these cells. We also mapped the CYP11A1 domains that confer structural changes of mitochondria. We searched for CYP11A1-interacting proteins and investigated the role of this interacting protein in shaping mitochondrial structure. Finally, we examined the effect of CYP11A1 overexpression on the amount of mitochondrial contact site and cristae organizing system. RESULTS: We found that CYP11A1 overexpression led to the formation of tubulovesicular cristae in mitochondria. We also identified the A'-helix located at amino acid #57-68 to be sufficient for membrane insertion and crista remodeling. We identified heat shock protein 60 (Hsp60) as the CYP11A1-interacting protein and showed that Hsp60 is required for CYP11A1 accumulation and crista remodeling. Finally, we found that the small MIC10 subcomplex of the mitochondrial contact site and cristae organizing system was reduced when CYP11A1 was overexpressed. CONCLUSIONS: CYP11A1 participates in the formation of tubulovesicular cristae in the mitochondria of steroidogenic cells. Its A'-helix is sufficient for the formation of tubulovesicular cristae and for protein integration into the membrane. CYP11A1 interacts with Hsp60, which is required for CYP11A1 accumulation. The accumulation of CYP11A1 leads to the reduction of MIC10 complex and changes mitochondrial structure.

Karyopherin Kap114p-mediated trans-repression controls ribosomal gene expression under saline stress.

Nuclear accessibility of transcription factors controls gene expression, co-regulated by Ran-dependent nuclear localization and a competitive regulatory network. Here, we reveal that nuclear import factor-facilitated transcriptional repression attenuates ribosome biogenesis under chronic salt stress. Kap114p, one of the karyopherin-ßs (Kap-ßs) that mediates nuclear import of yeast TATA-binding protein (yTBP), exhibits a yTBP-binding affinity four orders of magnitude greater than its counterparts and suppresses binding of yTBP with DNA. Our crystal structure of Kap114p reveals an extensively negatively charged concave surface, accounting for high-affinity basic-protein binding. KAP114 knockout in yeast leads to a high-salt growth defect, with transcriptomic analyses revealing that Kap114p modulates expression of genes associated with ribosomal biogenesis by suppressing yTBP binding to target promoters, a trans-repression mechanism we attribute to reduced nuclear Ran levels under salinity stress. Our findings reveal that Ran integrates the nuclear transport pathway and transcription regulatory network, allowing yeast to respond to environmental stresses.

Factors Associated With Frailty in Patients Undergoing Cardiac Surgery: A Longitudinal Study.

BACKGROUND: Frailty may increase the risk of complications and mortality in patients undergoing cardiac surgery. Few studies on frailty and its associated factors have been conducted in these patients. OBJECTIVE: The aim of this study was to explore frailty and related factors in patients undergoing cardiac surgery. METHODS: A total of 154 patients undergoing cardiac surgery in northern Taiwan were recruited using a longitudinal study design and interviewed using structured questionnaires assessing physical activity, anxiety and depression, and social support before surgery and at 1 month and 3 months after surgery. RESULTS: The prevalence of frailty in patients undergoing cardiac surgery was 16.2%, 20.5%, and 16.6% before surgery and at 1 month and 3 months after surgery, respectively. Frail and prefrail patients undergoing cardiac surgery were more likely to be unemployed, have gout, have a higher New York Heart Association class, have preoperative dysrhythmia, undergo cardiopulmonary bypass, have a lower functional ability, have a higher European System for Cardiac Operative Risk Evaluation score, have a longer anesthesia time, have longer endotracheal tube and extracorporeal circulation times, have longer intensive care unit and hospital stays, have lower hemoglobin and albumin levels, have higher anxiety and depression levels, and have lower Mini-Mental State Examination scores. The significant predictors of prefrailty and frailty included unemployment, the presence of gout, higher New York Heart Association classes, less independence in activities of daily living, lower hemoglobin levels, and higher levels of depression. CONCLUSIONS: Frailty was associated with patients' functional status, perioperative conditions and psychosocial factors. Preoperative assessments of frailty and appropriate interventions are needed to improve frailty in patients undergoing cardiac surgery.

Short-term international dental experiences in undergraduate dental students at an Australian university.

INTRODUCTION: Learning through international experience has been well documented in USA literature, yet not well explored in Australia. The aim of this study is to understand Australian dental students' experiences and perceptions of international dental experience by: (i) capturing past participation; (ii) identifying potential interests for participants who have not been on a trip and (iii) ascertaining perceived benefits of the trip/s. MATERIALS AND METHODS: A 19 question paper survey was distributed to 310 eligible undergraduate dental students of The University of Queensland (UQ). An online counterpart survey was created via Google Forms and promoted on relevant Facebook groups targeted at current dental students of UQ. RESULTS: A total of 203 of the 310 eligible UQ undergraduate dental students responded to the distributed paper and electronic surveys making the response rate 65.5%. A total of 13 undergraduate respondents participated in an international experience trip. The majority of respondents who had not participated in an international dental experience trip were interested in participating in one. Education (84.6%) and improved dental skills (84.6%) were the most reported perceived benefits by the students who had participated in an international dental experience. CONCLUSION: A majority of respondents were interested in participating in international dental experiences. Despite this, only 1 in 15 respondents had participated in an international dental experience. Further exploration of this concept is needed; however, dental schools within Australia could potentially explore integration of international short-term dental experiences into formal curricula in the future.

An H3K36 methylation-engaging Tudor motif of polycomb-like proteins mediates PRC2 complex targeting.

Polycomb repressive complex 2 (PRC2) regulates pluripotency, differentiation, and tumorigenesis through catalysis of histone H3 lysine 27 trimethylation (H3K27me3) on chromatin. However, the mechanisms that underlie PRC2 recruitment and spreading on chromatin remain unclear. Here we report that histone H3 lysine 36 trimethylation (H3K36me3) binding activity is harbored in the Tudor motifs of PRC2-associated polycomb-like (PCL) proteins PHF1/PCL1 and PHF19/PCL3. Ectopically expressed PHF1 induced Tudor-dependent stabilization of PRC2 complexes on bulk chromatin and mediated spreading of PRC2 and H3K27me3 into H3K36me3-containing chromatin regions. In murine pluripotent stem cells, we identified coexistence of H3K36me3, H3K27me3, and PHF19/PCL3 at a subset of poised developmental genes and demonstrated that PHF19/PCL3 Tudor function is required for optimal H3K27me3 and repression of these loci. Collectively, our data suggest that PCL recognition of H3K36me3 promotes intrusion of PRC2 complexes into active chromatin regions to promote gene silencing and modulate the chromatin landscape during development.

A TAF4 coactivator function for E proteins that involves enhanced TFIID binding.

The multisubunit TFIID plays a direct role in transcription initiation by binding to core promoter elements and directing preinitiation complex assembly. Although TFIID may also function as a coactivator through direct interactions with promoter-bound activators, mechanistic aspects of this poorly defined function remain unclear. Here, biochemical studies show a direct TFIID-E-protein interaction that (1) is mediated through interaction of a novel E-protein activation domain (activation domain 3 [AD3]) with the TAF homology (TAFH) domain of TAF4, (2) is critical for activation of a natural target gene by an E protein, and (3) mechanistically acts by enhancing TFIID binding to the core promoter. Complementary assays establish a gene-specific role for the TAFH domain in TFIID recruitment and activation of a large subset of genes in vivo. These results firmly establish TAF4 as a bona fide E-protein coactivator as well as a mechanism involving facilitated TFIID binding through direct interaction with an E-protein activation domain.

Andrographolide and its fluorescent derivative inhibit the main proteases of 2019-nCoV and SARS-CoV through covalent linkage.

The coronavirus disease 2019 (COVID-19) pandemic caused by 2019 novel coronavirus (2019-nCoV) has been a crisis of global health, whereas the effective vaccines against 2019-nCoV are still under development. Alternatively, utilization of old drugs or available medicine that can suppress the viral activity or replication may provide an urgent solution to suppress the rapid spread of 2019-nCoV. Andrographolide is a highly abundant natural product of the medicinal plant, Andrographis paniculata, which has been clinically used for inflammatory diseases and anti-viral therapy. We herein demonstrate that both andrographolide and its fluorescent derivative, the nitrobenzoxadiazole-conjugated andrographolide (Andro- NBD), suppressed the main protease (Mpro) activities of 2019-nCoV and severe acute respiratory syndrome coronavirus (SARS-CoV). Moreover, Andro-NBD was shown to covalently link its fluorescence to these proteases. Further mass spectrometry (MS) analysis suggests that andrographolide formed a covalent bond with the active site Cys145 of either 2019-nCoV Mpro or SARS-CoV Mpro. Consistently, molecular modeling analysis supported the docking of andrographolide within the catalytic pockets of both viral Mpros. Considering that andrographolide is used in clinical practice with acceptable safety and its diverse pharmacological activities that could be beneficial for attenuating COVID-19 symptoms, extensive investigation of andrographolide on the suppression of 2019-nCoV as well as its application in COVID-19 therapy is suggested.

A stable transcription factor complex nucleated by oligomeric AML1-ETO controls leukaemogenesis.

Transcription factors are frequently altered in leukaemia through chromosomal translocation, mutation or aberrant expression. AML1-ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukaemia, is a transcription factor implicated in both gene repression and activation. AML1-ETO oligomerization, mediated by the NHR2 domain, is critical for leukaemogenesis, making it important to identify co-regulatory factors that 'read' the NHR2 oligomerization and contribute to leukaemogenesis. Here we show that, in human leukaemic cells, AML1-ETO resides in and functions through a stable AML1-ETO-containing transcription factor complex (AETFC) that contains several haematopoietic transcription (co)factors. These AETFC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, co-localize genome wide, cooperatively regulate gene expression, and contribute to leukaemogenesis. Within the AETFC complex, AML1-ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2-N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1-ETO-induced haematopoietic stem/progenitor cell self-renewal and leukaemogenesis. These results reveal new mechanisms of action of AML1-ETO, and provide a potential therapeutic target in t(8;21)-positive acute myeloid leukaemia.